1. Field of the Invention
The present invention is related to a fluorescence detecting method and a fluorescence detecting apparatus that utilize surface plasmon. More specifically, the present invention is related to a fluorescence detecting method and a fluorescence detecting apparatus that utilizes a correcting mechanism to correct detected intensities of fluorescence.
2. Description of the Related Art
Conventionally, detecting methods that utilize totally reflected illumination are being focused on, in biological measurements for detecting proteins, DNA, and the like. These detection methods detect the presence or the amount of a detection target substance, by analyzing optical interactions such as scattering, absorption, and light emission, between light that leaks out when a measuring light beam is totally reflected at an interface between materials having different refractive indices, that is, evanescent waves, and the detection target substance, which is included in a sample, or labels attached to the detection target substance.
An example of such a detecting method is a fluorescence detecting method that utilizes fluorescent labels (refer to Margarida M. L. M. Vareiro et al., “Surface Plasmon Fluorescence Measurements of Human Chorionic Gonadotrophin: Role of Antibody Orientation in Obtaining Enhanced Sensitivity and Limit of Detection”, Analytical Chemistry, Vol. 77, No. 8, pp. 2426-2431, 2005.)
With recent advances in the performance of photodetectors, such as cooled CCD's, fluorometry has become indispensable in biological research. In addition, fluorescent pigments having fluorescence quantum yields that exceed 0.2, which is the standard for practical use, such as FITC (fluorescence: 525 nm, fluorescence quantum yield: 0.6) and Cy5 (fluorescence: 680 nm, fluorescence quantum yield: 0.3) have been developed as fluorescent labeling materials and are being widely used. Further, high sensitivity detection on the order of 1 pM and less is being realized, by amplifying fluorescence signals employing electric field enhancing fields due to surface plasmon.
The principles of the aforementioned fluorescence detecting method will be explained with reference to FIG. 7.
FIG. 7 is a conceptual diagram that illustrates a fluorescence detecting apparatus. For the sake of convenience in the explanation, the dimensions of each component are not drawn to actual scale.
The fluorescence detecting apparatus illustrated in FIG. 7 is equipped with: a sensor chip 10 constituted by a dielectric plate 11 and a metal film 12 provided at a predetermined region on one surface of the dielectric plate; an excitation light outputting optical system 20 that outputs an excitation light beam L0 at an incident angle that satisfies conditions for total reflection at the interface between the dielectric plate 11 and the metal film 12 from the side of the sensor chip 10 opposite the surface on which the metal film 12 is formed; and a photodetector 30 that detects fluorescence L0 generated by fluorescent labels F, which are attached to a detection target substance A, in the case that the detection target substance A having the fluorescent labels F attached thereto are present in a sample S in contact with the metal film 12.
In this fluorescence detecting apparatus, the excitation light beam L0 is output from the excitation light outputting optical system 20 and enters the interface between the dielectric plate 11 and the metal film 12 at a specific incident angle greater than or equal to a total reflection angle. Thereby, evanescent waves leak into the sample S on the metal film 12, and surface plasmon within the metal film 12 are excited by the evanescent waves. The evanescent waves and the surface plasmon cause an electric field enhancing field Ew that exhibits an electric field enhancing effect to be formed locally on the surface of the metal film 12.
A case will be considered in which antigens A are detected from within a sample S that includes the antigens A as a detection target substance. The metal film 12 is modified with primary antibodies B1 that specifically bind with the antigens A. The sample S is caused to flow into a sample holding section 13, and then secondary antibodies B2, which are modified with fluorescent labels F and that also specifically bind with the antigens A, are caused to flow into the sample holding section 13. At this time, the fluorescent labels F are immobilized to the metal film 12 via the specific bonds of the primary antibodies B1, the antigens A, and the secondary antibodies B2.
In the case described above, the fluorescent labels F are present within the electric field enhancing field Ew, and the fluorescent labels F are excited and caused to emit fluorescence Lf. Accordingly, the antigens A can be detected by detecting the fluorescence Lf. Note that the presence of the fluorescent labels F is actually directly confirmed by the detection of fluorescence. However, it is considered that the fluorescent labels F would not be immobilized onto the metal film 12 unless the antigens A are present. Therefore, the binding between the secondary antibodies B2 and the antigens A, that is, the presence of the antigens A, is indirectly confirmed by confirming the presence of the fluorescent labels F.
However, in the aforementioned fluorescent detecting method, there are many factors that contribute to fluctuations in the intensity of the electric field enhancing field generated by surface plasmon. Therefore, it is difficult to completely uniformize all of the factors for each measurement. This causes a problem that detected intensities of fluorescence fluctuate, and that reproducibility is poor, even if the amounts of detection target substances within samples are the same. These factors include: margins of error in the shapes and settings of sensor chips; shifting in the incident angle of excitation light beams caused by human error such as margins of errors in sensing and margins of error in setup of the optical system; and shifting in surface plasmon generating conditions due to physical factors such as the refractive indices of samples, the refractive indices of the sensor chips, irregularities on the surfaces of the sensor chips, the thicknesses of the metal films, and the densities of the metal films. In addition, changes in environmental temperatures during measurement may also lead to shifting in the aforementioned conditions, and therefore must be considered as well.
Variations in the aforementioned factors can be suppressed by selecting or exchanging components for optimal components for each measurement. However, extreme amounts of trouble and cost will be incurred if variations in all of the factors are suppressed by selecting or exchanging components.